WO2019222871A1 - 一种仿生肠-肝器官芯片及其制备方法和应用 - Google Patents

一种仿生肠-肝器官芯片及其制备方法和应用 Download PDF

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WO2019222871A1
WO2019222871A1 PCT/CN2018/087599 CN2018087599W WO2019222871A1 WO 2019222871 A1 WO2019222871 A1 WO 2019222871A1 CN 2018087599 W CN2018087599 W CN 2018087599W WO 2019222871 A1 WO2019222871 A1 WO 2019222871A1
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fluid channel
chip
intestinal
photoresist
bionic
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PCT/CN2018/087599
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English (en)
French (fr)
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魏文博
陈娟娟
邹远强
肖亮
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深圳华大生命科学研究院
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Priority to CN201880092380.2A priority Critical patent/CN111971384B/zh
Priority to PCT/CN2018/087599 priority patent/WO2019222871A1/zh
Publication of WO2019222871A1 publication Critical patent/WO2019222871A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present application relates to the field of organ bionics technology, and in particular, to a bionic intestinal-liver organ chip based on microfluidic technology and its application.
  • Microfluidics as one of the important frontier science and technology in the 21st century, provides an important platform for simulating human metabolic models in vitro. It is mainly based on micro-nano processing technology, formed by a network of micron-scale channels, and controls the fluid through the entire system, which can realize the routine functions of biology and chemistry laboratories. Because it has micron-sized components that match the cell size, it can perform a variety of cell culture and fluid stimulation in the microchannel of the chip, and build a three-dimensional microenvironment that is close to the physiological environment and has the characteristics of space-time resolution. Important technologies for screening, toxicology, and biomedical research.
  • Human Organs-on-a-chip is a new cutting-edge interdisciplinary technology developed in recent years. It is a kind of micro-fabrication chip that can simulate human organs using micro-processing technology. The main function of the bionic system. Compared with the traditional two-dimensional static cell culture technology, the cells cultured on the chip have a three-dimensional structure and a spatial distribution structure of a variety of cells. More importantly, the organ chip can provide a dynamic microenvironment for the cells, which is unmatched by traditional methods. . In addition, most of the cells in the chip are based on human-derived cells, which can greatly reduce the interspecific differences produced by animal models. The development of organ chips will help drug development and disease research.
  • the current intestinal-liver chip flow channel is in the lower layer, and different cells are seeded in a commercial transwell, and combined with the lower layer fluid to form a multi-organ combination.
  • the 2-organ-chip of TISSUSE is a two-dimensional cell. Culture, and cells cultured in transwell are in a relatively static environment.
  • the present application provides a bionic intestinal-liver organ chip that perfects intestinal-hepatocytes through the dynamic cultivation function of three-dimensional structure, and a preparation method and application thereof.
  • an embodiment provides a bionic intestinal-liver organ chip based on microfluidic technology, which has a fluid channel, and a porous membrane is provided in the fluid channel.
  • the porous membrane separates the fluid channel into an upper fluid channel and
  • the lower layer fluid channel is provided with a plurality of groove structures at the bottom of the lower layer fluid channel.
  • an embodiment provides a method for preparing a bionic intestinal-liver organ chip, including the following steps:
  • Seal assembly seal the porous membrane between the upper chip and the lower chip to form a bionic intestine-liver organ chip
  • the lower surface of the upper chip has an open upper fluid channel
  • the upper surface of the upper chip has an open lower fluid channel
  • a plurality of groove structures are provided at the bottom of the lower fluid channel.
  • an embodiment provides a method for preparing a bionic intestinal-liver digestive system, using the bionic intestinal-liver organ chip described above to perform a bionic intestinal-liver organ chip.
  • intestinal cells are inoculated in the upper channel, and intestinal epithelial tissues with metabolism and absorption functions are formed on the porous membrane.
  • Hepatocytes are inoculated in the lower channel. Because there are several groove structures at the bottom of the lower fluid channel, after inoculating liver cells, hepatocytes will gather in the groove structure to form hepatocyte spheres. Cells are closer to human liver cells and have more complete liver functions. The use of this chip can more accurately simulate the circulation system of the intestine and liver, which is conducive to research in the fields of pharmacokinetics, drug screening, food safety and other research fields.
  • FIG. 1 is a schematic structural diagram of a bionic intestinal-liver organ chip in the first embodiment
  • Example 2 is a flowchart of a method for preparing a bionic intestinal-liver organ chip in Example 2;
  • Embodiment 3 is a flowchart of preparing an upper-layer chip in Embodiment 2;
  • Example 6 is a flowchart of preparing a bionic intestinal-liver digestive system in Example 3.
  • This embodiment provides a bionic intestinal-hepatic organ chip.
  • the bionic intestinal-hepatic organ chip is used to cultivate intestinal cells and liver cells, and simulates the interaction between intestinal cells and liver cells and the enterohepatic circulation system, which is conducive to drug generation. Research work in the fields of kinetics, drug screening, food safety, etc.
  • the bionic intestinal-liver organ chip in this embodiment is provided with a fluid channel, and a porous membrane is provided between the fluid channels to divide the fluid channel into upper and lower channels.
  • the bionic bowel-liver organ chip includes an upper chip 110 and a lower chip 120.
  • the lower surface of the upper chip 110 has an open upper fluid channel
  • the upper surface of the lower chip 120 has an open lower fluid channel.
  • the porous membrane 130 is sealed on the upper chip 110.
  • the porous film 130 and the upper layer chip 110 surround the upper layer fluid channel 111
  • the porous film 130 and the lower layer chip 120 surround the lower layer fluid channel 121.
  • a plurality of groove structures 122 are arranged at the bottom of the lower fluid passage 121, and the plurality of groove structures 122 are arranged in an array.
  • the groove structures 122 are ladder-shaped groove structures.
  • the groove structures 122 are used to inoculate liver cells, so that liver cells It will gather in the groove structure 122 to form a hepatocyte sphere, which is closer to the human liver and has a more perfect liver function.
  • the volume ratio between the lower fluid channel 121 and the upper fluid channel 111 is about 3: 4, which is similar to the real volume ratio of the intestine and liver, making the simulated intestinal cell and liver cell interaction more accurate.
  • the upper-layer chip 110 and the lower-layer chip 120 are made of PDMS, SEBS, PMMA, PC, or PE.
  • the porous membrane 130 is covered with through holes having a pore diameter of 0.22-10 micrometers. The through holes on the porous membrane 130 are used to conduct the upper fluid channel 111 and the lower fluid channel 121. Intestinal cells are seeded in the porous membrane 130 in the upper fluid channel 111. on.
  • the porous film 130 is a polymer material film or a biomaterial film.
  • the molecular material film includes at least a PDMS film, a PC film, a nitrocellulose film, and a PET film.
  • the biomaterial film includes at least an alginic acid film, a chitosan film, a collagen film, and a gelatin film. .
  • the length of the upper fluid passage 111 and the lower fluid passage 121 are 10-15 mm and the width is 0.5-1.5 mm; the height of the upper fluid passage 111 is 0.1-0.5 mm, and the height of the lower fluid passage 121 is 0.1-0.2 mm. .
  • the depth of the groove structure 122 is 0.2-0.5 mm, the length of the upper diameter side of the groove structure 122 is 0.4-0.6 mm, and the length of the lower diameter side of the groove structure 122 is 0.15-0.2 mm.
  • the bionic intestinal-hepatic organ chip provided in this embodiment, because the intestinal cells in the upper fluid channel 111 are cultured on a porous membrane, and the perfusion culture will simulate the dynamic microenvironment of the intestine, so that the intestinal tissue formed in the chip has metabolism and Absorptive function; because several groove structures 122 are provided at the bottom of the lower fluid channel 121, after inoculating liver cells, hepatocytes will gather in the groove structure 122 to form hepatocyte spheres, and hepatocytes are dynamically cultivated through a three-dimensional structure It is closer to human hepatocytes and has more perfect liver function.
  • the bionic intestinal-liver organ chip can more accurately simulate the intestinal interaction with the liver and the enterohepatic circulation system, which is conducive to pharmacokinetics and drug screening. And food safety research.
  • This embodiment provides a method for preparing a bionic intestine-liver organ chip.
  • This preparation method mainly uses soft photolithography to prepare the bionic intestine-liver organ chip in the first embodiment.
  • the method for preparing the bionic intestinal-liver organ chip of this embodiment mainly includes the following steps:
  • step 300 a porous membrane is sealed between the upper chip and the lower chip to form a bionic intestine-liver organ chip.
  • Arrayed through holes are distributed on the produced porous membrane.
  • the porous membrane and the upper chip surround the upper fluid channel
  • the porous membrane and the lower chip surround the lower fluid channel
  • the bottom of the lower fluid channel also has an array of terrace-shaped grooves.
  • a bionic intestinal-liver organ chip is divided into two chips, an upper chip and a lower chip, and a porous membrane is sealed between the upper chip and the lower chip to form a three-layer structure chip with upper and lower circulation channels.
  • step S100 (preparing an upper-layer chip) includes the following steps:
  • S101 spin-coat a photoresist on a substrate surface of a glass or silicon wafer, and perform pre-baking;
  • SU-8 photoresist is preferred.
  • SU-8 photoresist is an epoxy-type, near-ultraviolet negative photoresist.
  • SU-8 photoresist has a low light absorption rate in the near-ultraviolet range. , So that it has better exposure uniformity in the thickness of the photoresist, and can obtain a structure with a nearly vertical edge of the pattern.
  • the mechanism of SU-8 photoresist photolithography is as follows: the photoinitiator in the photoresist absorbs photons and chemically reacts to produce a strong acid, which acts as an acid catalyst to promote the crosslinking reaction during the pre-baking process. Strong acid is only generated in the photoresist in the exposed area, so the cross-linking reaction only occurs in the exposed area. The cross-linking reaction does not occur in the unexposed area, and the photoresist is insoluble in the developing solution after the cross-linking reaction. The photoresist is soluble in the developing solution without cross-linking reaction, so the developed photoresist forms a pattern opposite to the mask pattern.
  • the thickness of the spin-coated SU-8 photoresist is 300 microns, which corresponds to the height of the upper fluid channel.
  • the pre-baking temperature was 95 ° C and the time was 4 hours.
  • the mask has a pattern of the upper fluid channel structure.
  • the mask is used to block ultraviolet light, so that the pattern on the mask is copied to the SU-8 photoresist.
  • Ultraviolet light is irradiated onto the SU-8 photoresist through the pattern on the mask.
  • the exposed area of the SU-8 photoresist will be crosslinked, and the crosslinked area will not be soluble in the developer.
  • the light sources in this embodiment are all ultraviolet light sources, and are used to emit ultraviolet light for exposure.
  • the post-baking temperature is 95 ° C and the time is 10-30 minutes.
  • the unexposed SU-8 photoresist is removed by a developing solution of ethyl lactate, and the developed SU-8 photoresist forms a pattern opposite to the mask, and then the film is strengthened.
  • the temperature of the film is 180 ° C for 2 hours.
  • S105 Prepare an upper chip by using a template having an upper fluid channel structure.
  • an upper layer chip of PDMS material is prepared by using a template with an upper layer fluid channel structure, and entrances are respectively made at the two ends of the upper layer fluid channel and the corresponding positions of the two ends of the lower layer fluid channel.
  • the upper chip can also be made of other materials, such as SEBS, PMMA, PC or PE.
  • the lower surface of the manufactured upper chip has an open upper fluid channel structure.
  • step S200 (the preparation method of the lower-layer chip) includes the following steps:
  • S201 spin-coat a photoresist on the substrate surface of a glass or silicon wafer, and perform the first pre-baking
  • a SU-8 photoresist with a thickness of 100 micrometers is spin-coated on a glass or silicon wafer. This thickness corresponds to the height of the lower fluid channel.
  • the first pre-baking temperature is 95 ° C and the time is 2 hours.
  • the SU-8 photoresist in the exposed area undergoes a crosslinking reaction and is insoluble in the developing solution.
  • S204 Remove the mask, spin-coat the photoresist on the exposed photoresist, and perform the second pre-baking;
  • SU-8 photoresist is spin-coated on the SU-8 photoresist.
  • the thickness of the SU-8 photoresist is 200-500 microns, for example, the thickness is 300 microns.
  • the thickness of the SU-8 photoresist Corresponds to the groove depth at the bottom of the lower fluid channel.
  • the temperature of the second pre-baking is 95 ° C, and the time is 4-8 hours, for example, 8 hours.
  • S205 Fix a mask having a circular pattern corresponding to the groove in the lower fluid channel on the surface of the substrate with two layers of photoresist, and the circular pattern is located in the exposure area of the first exposure;
  • the square pattern on the mask is an array distribution corresponding to the bottom surface of the groove at the bottom of the lower fluid channel, and is a template for preparing the groove at the bottom of the lower fluid channel.
  • the tilt angle of the platform is 30-60 degrees.
  • the second exposure is divided into 8 times.
  • the platform rotates 45 degrees after each storage. .
  • Ethyl lactate developer is used to remove the unexposed SU-8 photoresist to form a SU-8 photoresist template with a lower fluid channel structure.
  • a lower-layer chip made of PDMS material is prepared by using a template with a lower-layer fluid channel structure.
  • the lower-layer chip may also be made of other materials, such as SEBS, PMMA, PC, or PE.
  • the upper surface of the prepared lower chip has an open lower fluid channel structure, and the volume ratio of the lower fluid channel structure of the lower chip to the upper fluid channel structure of the upper chip is 3: 4.
  • step S300 (sealing assembly) includes the following steps:
  • the porous membrane is fixed on the upper chip by means of hot pressing or bonding.
  • the lower layer chip is aligned with the upper layer chip with a porous membrane, and fixed by heat pressing or bonding.
  • the lower layer chip is sealed on the other side of the porous membrane.
  • the porous membrane is a PC membrane, and may also be a high-resolution material or a biomaterial membrane.
  • the sealing of the PC porous film and the upper chip can be completed during the preparation of the upper chip, that is, after the sidewall of the upper chip prepared by the template is adhered to the uncured PDMS of the glass, the porous film is adhered to Bake on the sidewall of the upper chip at 80 ° C for 2 hours to cure the PDMS. At the same time, the preparation of the upper chip and sealing of the porous film are completed. Then, the upper chip and the lower chip are sealed by plasma treatment. Come together.
  • the method for preparing a bionic intestine-liver organ chip mainly uses soft photolithography to prepare a bionic intestine-liver organ chip.
  • the preparation efficiency is high, and the SU-8 photoresist is used for template preparation, which can produce structural accuracy. High chip.
  • This embodiment provides a method for preparing a bionic intestine-liver digestive system, which is performed using the bionic intestine-liver organ chip described in the first embodiment. This method is an application of the bionic intestine-liver organ chip.
  • intestine and liver tissues are respectively constructed in the upper and lower channels of the chip, as shown in FIG. 6, and specifically include the following steps:
  • S401 sterilize the bionic intestinal-liver organ chip
  • the bionic intestinal-liver organ chip described in Example 1 was sterilized with 70% alcohol and ultraviolet rays, respectively.
  • S402 Inject extracellular matrix solution into the upper fluid channel, modify the porous membrane, and clean the fluid channel after modification;
  • the intestinal cells (Caco-2) was inoculated in accordance with a concentration of 10 6 / ml into the upper layer of the fluid passage within the chip, still more 2 hours after the porous film grown with the modified cell adhesion;
  • S405 Inject the culture medium into the upper and lower fluid channels continuously at a certain flow rate. After several days of incubation, the intestinal cells mature and form a three-dimensional intestinal microtissue;
  • Intestinal cells (Caco-2) are perfused and cultured. After 5-7 days, the intestinal cells (Caco-2) will mature and form intestinal microtissue in the upper fluid channels in the chip;
  • the culture medium is continuously injected into the upper and lower fluid channels respectively at a certain flow rate. After several days of culture, the hepatocytes gathered in the groove structure will spontaneously form hepatocyte spheres.
  • the aggregated hepatocytes spontaneously formed hepatocyte globules.
  • Hepatocytes (HepG2) seeded in accordance with a concentration of 10 6 / ml into the lower fluid channel chip, stationary for 12 hours to allow HepG2 cell aggregation structure in the groove, and then the medium according to a certain flow rate were continuously injected into the upper layer and In the lower fluid channel, after 3-5 days of culture, the hepatocytes gathered in the groove structure will spontaneously form hepatocyte globules.
  • the functional intestinal micro-tissue and liver micro-tissue are formed in the chip at the same time, forming a bionic intestinal-liver digestive system, which can be applied to related research work.

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Abstract

一种基于微流控技术的仿生肠-肝器官芯片及其制备方法和应用，该仿生肠-肝器官芯片具有流体通道，在流体通道内设有多孔膜（130），多孔膜（130）将流体通道分隔成上层流体通道（111）和下层流体通道（121），下层流体通道（121）的底部设有若干个凹槽结构（122）。肠细胞接种于上层通道（111）内，并在多孔膜（130）上形成具有代谢与吸收功能的肠道上皮组织。肝细胞接种于下层通道（121）内，由于在下层流体通道（121）的底部设有若干个凹槽结构（122），接种肝细胞后，肝细胞会聚集在凹槽结构（122）内形成肝细胞球，通过三维动态培育出的肝细胞与人体肝细胞更为接近，具有更完善的肝功能。利用该芯片可更准确的模拟肠道与肝脏的循环体系，有利于药代动力学、药物筛选、食品安全等研究领域的研究工作。

Description

一种仿生肠-肝器官芯片及其制备方法和应用 技术领域
本申请涉及器官仿生技术领域，具体涉及一种基于微流控技术的仿生肠-肝器官芯片及其应用。
背景技术
微流控芯片技术(Microfluidics)作为21世纪重要前沿科学技术之一，为体外模拟人体代谢模型提供了一种重要平台。它主要以微纳加工技术为基础，由微米级通道形成网络，以可控流体贯穿整个系统，可实现生物学与化学实验室的常规功能。因其具有与细胞大小相匹配的微米尺寸构件，可在芯片微通道内进行多种细胞培养与流体刺激，构建与生理环境接近并具有时空分辨特点的三维微环境，已成为组织器官构建、药物筛选、毒理学以及生物医学研究的重要技术。现阶段微流控技术已成功应用于三维细胞共培养、细胞迁移、细胞分选、组织微环境与类器官构建等。其中，人体器官芯片(Organs-on-a-chip)是近几年发展起来的一种新兴前沿交叉学科技术，是一种利用微加工技术，在微流控芯片上制造出能够模拟人类器官的主要功能的仿生系统。与传统二维静态细胞培养技术相比，芯片内培养的细胞具有三维结构以及多种细胞的空间分布结构，更重要的是器官芯片可以为细胞提供动态的微环境，这是传统手段无法比拟的。此外，芯片内的细胞大部分都是基于人源的细胞，可以极大的降低动物模型所产生的种间差异。器官芯片的发展将有助于药物研发、疾病研究等。
目前，已有一些研究组和公司已经开发出多器官芯片，现有的肠-肝芯片流通通道在下层，将不同细胞接种在商品化的transwell里，并结合下层的流体形成多器官连用。
现有多器官芯片只是将不同组织细胞接种在不同区域内，没有考虑到不同组织的三维结构，会造成一些组织细胞功能的弱化或缺失，如TISSUSE公司的2-organ-chip，是二维细胞培养，并且培养在transwell中的细胞处于相对静态环境中。
发明内容
本申请提供一种通过三维结构动态培育功能完善肠-肝细胞的仿生肠-肝器官芯片及其制备方法和应用。
根据第一方面，一种实施例中提供一种基于微流控技术的仿生肠-肝器官芯片，具有流体通道，在流体通道内设有多孔膜，多孔膜将流体通道分隔成上层流体通道和下层流体通道，下层流体通道的底部设有若干个凹槽结构。
根据第二方面，一种实施例中提供一种仿生肠-肝器官芯片的制备方法，包括如下步骤：
制备上层芯片；
制备下层芯片；
封接组装：将多孔膜封接在上层芯片和下层芯片之间，形成仿生肠-肝器官芯片；
其中，上层芯片的下表面具有开放的上层流体通道，上层芯片的上表面具有开放的下层流体通道，下层流体通道的底部设有若干个凹槽结构。
根据第三方面，一种实施例中提供了一种制备仿生肠-肝消化系统的方法，利用上述的仿生肠-肝器官芯片进行仿生肠-肝器官芯片。
依据上述实施例的仿生肠-肝器官芯片及其制备方法和应用，肠细胞接种于上层通道内，并在多孔膜上形成具有代谢与吸收功能的肠道上皮组织。肝细胞接种于下层通道内，由于在下层流体通道的底部设有若干个凹槽结构，接种肝细胞后，肝细胞会聚集在凹槽结构内形成肝细胞球，通过三维结构动态培育出的肝细胞与人体肝细胞更为接近，具有更完善的肝功能。利用该芯片可更准确的模拟肠道与肝脏的循环体系，有利于药代动力学、药物筛选、食品安全等研究领域的研究工作。
附图说明
图1为实施例一中仿生肠-肝器官芯片的结构示意图；
图2为实施例二中仿生肠-肝器官芯片制备方法的流程图；
图3为实施例二中制备上层芯片的流程图；
图4为实施例二中制备下层芯片的流程图；
图5为实施例二中封接组装的流程图；
图6为实施例三中制备仿生肠-肝消化系统的流程图。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。
实施例一：
本实施例提供了一种仿生肠-肝器官芯片，本仿生肠-肝器官芯片用于培育肠细胞和肝细胞，并模拟肠细胞和肝细胞的相互作用以及肠肝循环体系，有利于药代动力学、药物筛选、食品安全等研究领域的研究工作。
如图1所示，本实施例的仿生肠-肝器官芯片内设有流体通道，并在流体通道之间设有多孔膜，将流体通道分为上下两层通道。仿生肠-肝器官芯片包括上层芯片110和下层芯片120，上层芯片110的下表面具有开放的上层流体通道，下层芯片120的上表面具有开放的下层流体通道；多孔膜130封接在上层芯片110和下层芯片120之间，多孔膜130和上层芯片110围合成上层流体通道111，多孔膜130和下层芯片120围合成下层流体通道121。
在下层流体通道121的底部设有若干个凹槽结构122，若干个凹槽结构122阵列分布设置，凹槽结构122为梯台形凹槽结构，凹槽结构122用于接种肝细胞，使得肝细胞会聚集在凹槽结构122内形成肝细胞球，与人体肝更接近，具有更完善的肝功能。
下层流体通道121和上层流体通道111之间的体积比约为3:4，与肠和肝的真实体积比相似，使得模拟的肠细胞和肝细胞的相互作用更准确。
本实施例中，上层芯片110和下层芯片120为PDMS、SEBS、PMMA、PC或PE等材质。多孔膜130上布满有0.22-10微米孔径的通孔，多孔膜130上的通孔用于导通上层流体通道111和下层流体通道121，肠细胞接种在上层流体通道111内的多孔膜130上。多孔膜130为高分子材料膜或生物材料膜，分子材料膜至少包括PDMS膜、PC膜、硝酸纤维膜和PET膜，生物材料膜至少包括海藻酸膜、壳聚糖膜、胶原膜和明胶膜。
具体的，上层流体通道111和下层流体通道121的长度为10-15毫米，宽度为0.5-1.5毫米；上层流体通道111的高度为0.1-0.5毫米，下层流体通道121的高度为0.1-0.2毫米。凹槽结构122的深度为0.2-0.5毫米，凹槽结构122的上口径边长为0.4-0.6毫米，凹槽结构122的下口径边长为0.15-0.2毫米。
本实施例提供的仿生肠-肝器官芯片，由于在上层流体通道111中的 肠细胞培养在多孔膜上，同时灌流培养会模拟肠道动态的微环境，使芯片内形成的肠组织具有代谢与吸收的功能；由于在下层流体通道121的底部设有若干个凹槽结构122，接种肝细胞后，肝细胞会聚集在凹槽结构122内形成肝细胞球，通过三维结构动态培育出的肝细胞与人体肝细胞更为接近，具有更完善的肝功能；利用该仿生肠-肝器官芯片可更准确的模拟肠道与肝脏的相互作用以及肠肝循环体系，有利于药代动力学、药物筛选、食品安全等研究领域的研究工作。
实施例二：
本实施例提供了一种仿生肠-肝器官芯片的制备方法，本制备方法主要采用软光刻技术制备上述实施例一中的仿生肠-肝器官芯片。
如图2所示，本实施例的仿生肠-肝器官芯片的制备方法主要包括如下步骤：
S100：制备上层芯片；
S200：制备下层芯片；
S300：封接组装。
步骤300中，将多孔膜封接在上层芯片和下层芯片之间，形成仿生肠-肝器官芯片。生产的多孔膜上分布有阵列的通孔，多孔膜与上层芯片围合成上层流体通道，多孔膜与下层芯片围合成下层流体通道，下层流体通道的底部还分布有阵列的梯台形凹槽。其中，步骤S100和S200无先后顺序，可先后制备，也可同时制备。
本制备方法，将仿生肠-肝器官芯片分开成上层芯片和下层芯片两个单体制备，再将多孔膜封接在上层芯片和下层芯片之间，形成具有上下层流通通道的三层结构芯片。
具体的，如图3所示，步骤S100(制备上层芯片)包括如下步骤：
S101：在玻璃或硅片的基底表面旋涂光刻胶，并进行前烘；
本实施例中优选SU-8光刻胶，SU-8光刻胶是一种环氧型的、近紫外光负光刻胶，SU-8光刻胶在近紫外光范围内光吸收率低，使得在光刻胶厚度上都具有较好的曝光均匀性，能够得到图形边缘近乎垂直的结构。
SU-8光刻胶光刻的机理如下：光刻胶中的光引发剂吸收光子发生了化学反应，生产一种强酸，其作用是前烘过程中作为酸催化剂促进交联反应。只有在曝光区域的光刻胶中才会产生强酸，故只有在曝光区域内才发生交联反应，未曝光的区域不发生交联反应，而光刻胶发生交联反应后不溶于显影液，而光刻胶未发生交联反应的溶于显影液，因此显影 后的光刻胶形成与掩膜图案相反的图形。
本步骤中，旋涂SU-8光刻胶的厚度为300微米，与上层流体通道的高度对应。前烘的温度为95℃，时间为4小时。
S102：将具有上层流体通道结构图案的掩膜固定于附有光刻胶的基底表面；
掩膜具有上层流体通道结构的图案，掩膜用于隔档紫外光，从而将掩膜上图案复制到SU-8光刻胶上。
S103：光源垂直照射掩膜进行曝光，并进行后烘；
紫外光穿过掩膜上的图案照射到SU-8光刻胶上，SU-8光刻胶被曝光的区域将发生交联，交联后的区域不溶于显影液。本实施例中的光源均为紫外光光源，用于发射紫外光进行曝光。
本步骤中，后烘的温度为95℃，时间为10-30分钟。
S104：自然冷却后，采用显影液去除未曝光的光刻胶，形成具有上层流体通道结构的模板，并进行坚膜；
本步骤中，通过乳酸乙酯显影液去除未曝光的SU-8光刻胶，显影后的SU-8光刻胶形成与掩膜相反的图形结构，再坚膜加固，坚膜的温度为180℃，时间为2小时。
S105：通过具有上层流体通道结构的模板制备上层芯片。
本步骤中，通过具有上层流体通道结构的模板制备出PDMS材质的上层芯片，并在上层流体通道两端位置与下层流体通道两端对应位置分别打出入口。
上层芯片也可为其他材质，例如SEBS、PMMA、PC或PE等。制成的上层芯片的下表面具有开放的上层流体通道结构。
如图4所示，步骤S200(下层芯片的制备方法)包括如下步骤：
S201：在玻璃或硅片的基底表面旋涂光刻胶，并进行第一次前烘；
本步骤中，在玻璃或硅片上旋涂厚度为100微米的SU-8光刻胶，此厚度与下层流体通道的高度对应，第一前烘的温度为95℃，时间为2小时。
S202：将具有下层流体通道图案的掩膜固定于附有光刻胶的基底表面；
S203：光源垂直照射掩膜进行第一次曝光；
第一次曝光后，曝光区域内SU-8光刻胶发生交联反应，不溶于显影液。
S204：去除掩膜，在经过曝光的光刻胶上再旋涂光刻胶，并进行第二次前烘；
第一曝光后，在SU-8光刻胶上旋涂SU-8光刻胶，SU-8光刻胶的厚度为200-500微米，例如厚度为300微米，SU-8光刻胶的厚度与下层流体通道底部的凹槽深度对应。第二次前烘的温度为95℃，时间为4-8小时，例如时间为8小时。
S205：将具有与下层流体通道内凹槽对应的圆形图案的掩膜固定于附有两层光刻胶的基底表面，并且圆形图案位于第一次曝光的曝光区域内；
掩膜上方形图案为阵列分布与下层流体通道底部的凹槽底面对应，用于制备下层流体通道底部的凹槽的模板。
S206：光源倾斜旋转照射吃进行第二次曝光，并进行后烘；
首先将固定有掩膜与两层光刻胶的基底置于可倾斜旋转的平台上，平台的倾斜角度为30-60度，第二次曝光分为8次，每次保管后平台旋转45度。
S207：自然冷却后，采用显影液去除未曝光的光刻胶，形成具有下层流体通道结构的模板，并进行坚膜；
采用乳酸乙酯显影液去除未曝光的SU-8光刻胶，形成具有下层流体通道结构的SU-8光刻胶模板。
S208：通过具有下层流体通道结构的模板制备下层芯片。
本步骤中，通过具有下层流体通道结构的模板制备出PDMS材质的下层芯片，下层芯片也可为其他材质，例如SEBS、PMMA、PC或PE等。制备成的下层芯片的上表面具有开放的下层流体通道结构，并且，下层芯片的下层流体通道结构与上层芯片的上层流体通道结构的体积比为3:4。
如图5所示，步骤S300(封接组装)包括如下步骤：
S301：将多孔膜封接在上层芯片上；
通过热压或键合等方式将多孔膜固定在上层芯片上。
S302：将下层芯片对准封接在多孔膜的另一侧，形成仿生肠-肝器官芯片。
将下层芯片与附有多孔膜的上层芯片对准，通过热压或键合等方式固定，下层芯片封接在多孔膜的另一侧。多孔膜为PC膜，也可为高分材料或生物材料膜。
在本实施例中，PC多孔膜与上层芯片的封接可在上层芯片的制备过程中完成，即当模板制得的上层芯片侧壁粘在玻璃的未固化PDMS上后，将多孔膜粘在上层芯片侧壁上，以80℃的温度烘烤2小时，使PDMS固化，同时完成上层芯片的制备和封接多孔膜，再利用等离子处理，将上层芯片与下层芯片通过键合的方式封接到一起。
本实施例提供的仿生肠-肝器官芯片的制备方法，主要采用软光刻技术制备仿生肠-肝器官芯片，制备效率高，并选用SU-8光刻胶进行模板制备，能够制备出结构精度高的芯片。
实施例三：
本实施例提供了一种制备仿生肠-肝消化系统的方法，利用实施例一中所述的仿生肠-肝器官芯片进行，本方法为对仿生肠-肝器官芯片的应用。
本实施例在芯片上下通道内分别构建肠与肝组织，如图6所示，具体包括如下步骤：
S401：对仿生肠-肝器官芯片进行灭菌处理；
分别利用70％的酒精和紫外线射线对实施例一中所述的仿生肠-肝器官芯片进行灭菌处理。
S402：将细胞外基质溶液注入到上层流体通道内，对多孔膜进行修饰，修饰后清洗流体通道；
将细胞外基质(I型胶原、基质胶matrigel等)溶液从上层芯片入口注入到通道内对多孔膜进行修饰，修饰后用无血清的培养基或PBS清洗通道；
S403：将PF-127溶液注入到下层流体通道内，对凹槽结构进行修饰，修饰后清洗流体通道；
将2％的PF-127溶液从下层芯片入口注入到通道内对凹槽结构进行修饰，修饰后用无血清的培养基或PBS清洗通道；
S404：将肠细胞悬液注入到上层流体通道内，静止一定时间，以使肠细胞黏附在修饰后的多孔膜上；
将肠细胞(Caco-2)按照10 6个/ml的浓度接种到芯片内的上层流体通道内，静止2小时以上使细胞黏附与修饰后的多孔膜上生长；
S405：将培养基按照一定的流速连续注入到上层和下层流体通道内，培育若干个天后，肠细胞分化成熟，形成三维肠道微组织；
对肠细胞(Caco-2)进行灌流培养，5-7天后肠细胞(Caco-2)将分 化成熟，在芯片内的上层流体通道形成肠道微组织；
S406：待肠组织分化成熟后，再将肝细胞悬液注入到下层流体通道内，静止一定时间，使肝细胞聚集在凹槽结构中；
S407：将培养基按照一定的流速分别连续注入上层和下层流体通道内，培养若干天后，聚集在凹槽结构中的肝细胞将自发形成肝细胞球。
培养若干天后，聚集的肝细胞自发形成肝细胞球。
将肝细胞(HepG2)按照10 6个/ml的浓度接种到芯片下层流体通道内，静止12小时，以使HepG2细胞聚集在凹槽结构中，再将培养基按照一定的流速分别连续注入上层和下层流体通道内，培养若3-5天后，聚集在凹槽结构中的肝细胞将自发形成肝细胞球。在芯片内同时形成功能化的肠道微组织与肝微组织，形成一个仿生肠-肝消化系统，可以应用于相关研究工作。
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (41)

  1. 一种仿生肠-肝器官芯片，具有流体通道，在所述流体通道内设有多孔膜，所述多孔膜将所述流体通道分隔成上层流体通道和下层流体通道，其特征在于，所述上层流体通道用于构建肠组织，下层流体通道用于构建肝组织，并且下层流体通道的底部设有若干个凹槽结构。
  2. 如权利要求1所述的仿生肠-肝器官芯片，其特征在于，若干个所述凹槽阵列分布在所述下层流体通道的底部。
  3. 如权利要求2所述的仿生肠-肝器官芯片，其特征在于，所述凹槽为梯台形凹槽。
  4. 如权利要求3所述的仿生肠-肝器官芯片，其特征在于，所述下层流体通道与上层流体通道的体积比为3:4。
  5. 如权利要求4所述的仿生肠-肝器官芯片，其特征在于，包括上层芯片和下层芯片，所述上层芯片的下表面具有开放的上层流体通道，所述下层芯片的上表面具有开放的下层流体通道；所述多孔膜封接在所述上层芯片和下层芯片之间，围合成所述上层流体通道和下层流体通道。
  6. 如权利要求5所述的仿生肠-肝器官芯片，其特征在于，所述上层芯片和下层芯片为PDMS、SEBS、PMMA、PC或PE材质。
  7. 如权利要求5所述的仿生肠-肝器官芯片，其特征在于，所述多孔膜为高分子材料膜或生物材料膜。
  8. 如权利要求6所述的仿生肠-肝器官芯片，其特征在于，所述高分子材料膜至少包括PDMS膜、PC膜、硝酸纤维膜和PET膜，所述生物材料膜至少包括海藻酸膜、壳聚糖膜、胶原膜和明胶膜。
  9. 如权利要求6所述的仿生肠-肝器官芯片，其特征在于，所述上层流体通道和下层流体通道的长度为10-15毫米，宽度为0.5-1.5毫米；上层流体通道的高度为0.1-0.5毫米，所述下层流体通道的高度为0.1-0.2毫米。
  10. 如权利要求6所述的仿生肠-肝器官芯片，其特征在于，所述凹槽的深度为0.2-0.5毫米，所述凹槽结构的上口径边长为0.4-0.6毫米，所述凹槽结构的下口径边长为0.15-0.2毫米。
  11. 如权利要求6所述的仿生肠-肝器官芯片，其特征在于，所述多孔膜上通孔的孔径为0.22-10微米。
  12. 一种仿生肠-肝器官芯片的制备方法，用于制备如权利要求1 至11中任一项所述的仿生肠-肝器官芯片，其特征在于，包括如下步骤：
    制备上层芯片；
    制备下层芯片；
    封接组装：将多孔膜封接在所述上层芯片和下层芯片之间，形成仿生肠-肝器官芯片；
    其中，所述上层芯片的下表面具有开放的上层流体通道，所述上层芯片的上表面具有开放的下层流体通道，所述下层流体通道的底部设有若干个凹槽结构。
  13. 如权利要求12所述的制备方法，其特征在于，所述制备上层芯片包括如下步骤：
    在玻璃或硅片的基底表面旋涂光刻胶，并进行前烘；
    将具有上层流体通道结构图案的掩膜固定于附有光刻胶的基底表面；
    光源垂直照射掩膜进行曝光，并进行后烘；
    自然冷却后，采用显影液去除未曝光的光刻胶，形成具有上层流体通道结构的模板，并进行坚膜；
    通过具有上层流体通道结构的模板制备上层芯片，并在上层流体通道两端位置与下层流体通道两端对应位置分别打出入口。
  14. 如权利要求13所述的制备方法，其特征在于，所述光刻胶的厚度为300微米。
  15. 如权利要求13所述的制备方法，其特征在于，所述前烘的温度为95℃，时间为4小时。
  16. 如权利要求13所述的制备方法，其特征在于，所述后烘的温度为95℃，时间为10-30分钟。
  17. 如权利要求13所述的制备方法，其特征在于，所述坚膜的温度为180℃，时间为2小时。
  18. 如权利要求12所述的制备方法，其特征在于，所述制备下层芯片包括如下步骤：
    在玻璃或硅片的基底表面旋涂光刻胶，并进行第一次前烘；
    将具有下层流体通道图案的掩膜固定于附有光刻胶的基底表面；
    光源垂直照射掩膜进行第一次曝光；
    去除掩膜，在经过曝光的光刻胶上再旋涂光刻胶，并进行第二次前烘；
    将具有与下层流体通道内凹槽对应的方形图案的掩膜固定于附有两层光刻胶的基底表面，并且所述方形图案位于第一次曝光的曝光区域内；
    将固定有掩膜与两层光刻胶的基底置于可倾斜旋转的平台上进行第二次曝光，并进行后烘；
    自然冷却后，采用显影液去除未曝光的光刻胶，形成具有下层流体通道结构的模板，并进行坚膜；
    通过具有下层流体通道与凹槽结构的模板制备下层芯片。
  19. 如权利要求13或18所述的制备方法，其特征在于，所述光刻胶为SU-8光刻胶，所述显影液为乳酸乙酯。
  20. 如权利要求13或18所述的制备方法，其特征在于，所述光源为紫外光光源。
  21. 如权利要求18所述的制备方法，其特征在于，所述光刻胶为SU-8光刻胶，所述上层芯片和下层芯片均为PDMS材质。
  22. 如权利要求18所述的制备方法，其特征在于，所述下层芯片上的下层流体通道与上层芯片上的上层流体通道的体积比为3:4。
  23. 如权利要求18所述的制备方法，其特征在于，所述光刻胶的厚度为100微米。
  24. 如权利要求18所述的制备方法，其特征在于，所述第一次前烘的温度为95℃，时间为2小时。
  25. 如权利要求18所述的制备方法，其特征在于，所述再次旋涂的光刻胶的厚度为200-500微米。
  26. 如权利要求18所述的制备方法，其特征在于，所述第二次前烘的温度为95℃，时间为4-8小时。
  27. 如权利要求18所述的制备方法，其特征在于，所述后烘的温度为95℃，时间为10-30分钟。
  28. 如权利要求18所述的制备方法，其特征在于，所述坚膜的温度为180℃，时间为2小时。
  29. 如权利要求12所述的制备方法，其特征在于，所述封接组装包括如下步骤：
    将多孔膜封接在上层芯片上；
    将下层芯片对准封接在多孔膜的另一侧，形成仿生肠-肝器官芯片。
  30. 如权利要求29所述的制备方法，其特征在于，所述多孔膜与上层芯片和下层芯片通过热压或键合的方式封接在一起。
  31. 一种制备仿生肠-肝消化系统的方法，其特征在于，利用权利要求1至11中任一项所述的仿生肠-肝器官芯片进行。
  32. 如权利要求31所述的方法，其特征在于，包括如下步骤：
    对如权利要求1至11中任一项所述的仿生肠-肝器官芯片进行灭菌处理；
    将细胞外基质溶液注入到上层流体通道内，对多孔膜进行修饰，修饰后清洗流体通道；
    将PF-127溶液注入到下层流体通道内，对凹槽结构进行修饰，修饰后清洗流体通道；
    将肠细胞悬液注入到上层流体通道内，静止一定时间，以使肠细胞黏附在修饰后的多孔膜上；
    将培养基按照一定的流速连续注入到上层和下层流体通道内，培育若干天后，肠细胞分化成熟，形成三维肠道微组织；
    待肠组织分化成熟后，再将肝细胞悬液注入到下层流体通道内，静止一定时间，使肝细胞在凹槽结构内聚集；
    将培养基按照一定的流速分别连续注入上层和下层流体通道内，培养若干天后，聚集在凹槽结构中的肝细胞将自发形成肝细胞球。
  33. 如权利要求32所述的方法，其特征在于，采用浓度为70％的酒精和紫外线对仿生肠-肝器官芯片进行灭菌处理。
  34. 如权利要求32所述的方法，其特征在于，所述细胞外基质溶液为I型胶原或基质胶matrigel溶液。
  35. 如权利要求32所述的方法，其特征在于，修饰后采用无血清的培养基或PBS清洗上层流体通道和下层流体通道。
  36. 如权利要求32所述的方法，其特征在于，所述PF-127溶液的浓度为2％。
  37. 如权利要求32所述的方法，其特征在于，所述肠细胞悬液和肝细胞悬液的浓度均为10 6个/ml。
  38. 如权利要求32所述的方法，其特征在于，所述肠细胞悬液注入到流体通道内后，静止两小时以上。
  39. 如权利要求32所述的方法，其特征在于，将培养基按照一定的流速连续注入到上层和下层流体通道内，，培育5-7天。
  40. 如权利要求32所述的方法，其特征在于，所述肝细胞悬液注入下层流体通道后，先静止12小时培育，此时上层流体通道继续进行 灌流培养，或者静止培养。
  41. 如权利要求32所述的方法，其特征在于，再将培养基按照一定的流速分别连续注入上层和下层流体通道内，培养若干天后，聚集在凹槽结构中的肝细胞将自发形成肝细胞球。
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